Cyclotides are a topologically unique family of plant proteins that are exceptionally stable (Craik, D. J., et al., (1999) J. Mol. Biol. 294, 1327-1336). They comprise ˜30 amino acids arranged in a head-to-tail cyclized peptide backbone that additionally is restrained by a cystine knot motif associated with six conserved cysteine residues. The cystine knot (Pallaghy, P. K., Nielsen, K. J., Craik, D. J. & Norton, R. S. (1994) Protein Sci. 3, 1833-1839) is built from two disulfide bonds and their connecting backbone segments forming an internal ring in the structure that is threaded by the third disulfide bond to form an interlocking and cross braced structure (FIG. 1). Superimposed on this cystine knot core motif are a well-defined β-sheet and a series of turns displaying short surface-exposed loops.
Cyclotides express a diversity of peptide sequences within their backbone loops and have a broad range of biological activities, including uterotonic (Gran, L. (1970) Medd. Nor. Farm. Selsk. 12, 173-180), anti-HIV (Gustafson, K. R., Sowder, R. C. I., Henderson, L. E., Parsons, I. C., Kashman, Y., Cardellina, J. H. I., McMahon, J. B., Buckheit, R. W. J., Pannell, L. K. & Boyd, M. R. (1994) J. Am. Chem. Soc. 116, 9337-9338), antimicrobial (Tam, J. P., Lu, Y. A., Yang, J. L. & Chiu, K. W. (1999) Proceedings of the National Academy of Sciences of the United States of America 96, 8913-8918), and anticancer activities (Svångard, E., Burman, R., Gunasekera, S., Lovborg, H., Gullbo, J. & Göransson, U. (2007) J Nat Prod 70, 643-7). They are thus of great interest for pharmaceutical applications. Some plants from which they are derived are used in indigenous medicines, including kalata-kalata, a tea from the plant Oldenlandia affinis that is used for accelerating childbirth in Africa that contains the prototypic cyclotide kalata B1 (Gran, L. (1973) Lloydia 36, 174-178). This ethnobotanical use and more recent biophysical studies (Colgrave, M. L. & Craik, D. J. (2004) Biochemistry 43, 5965-5975) illustrate the remarkable stability of cyclotides, i.e., they survive boiling and ingestion, observations unprecedented for conventional peptides. Their exceptional stability means that they have attracted attention as potential templates in peptide-based drug design applications (Craik, D. J. (2006) Science 311, 1563-1564). In particular, the grafting of bioactive peptide sequences into a cyclotide framework offers the promise of a new approach to stabilize peptide-based therapeutics, thereby overcoming one of the major limitations on the use of peptides as drugs. Chemical (Daly, N. L., Love, S., Alewood, P. F. & Craik, D. J. (1999) Biochemistry 38, 10606-14; Tam, J. P. & Lu, Y.-A. (1998) Protein Sci. 7, 1583-1592), chemo-enzymatic (Thongyoo, P., Roque-Rosell, N., Leatherbarrow, R. J. & Tate, E. W. (2008) Org Biomol Chem 6, 1462-1470), and recombinant (Camarero, J. A., Kimura, R. H., Woo, Y.-H., Shekhtman, A. & Cantor, J. (2007) ChemBioChem 8, 1363-1366) approaches to the synthesis of cyclotides have been developed, thus facilitating these pharmaceutical applications. See also WO 01/27147 to Craik, et al, and WO 01/34829 to Craik, et al., each incorporated herein by reference.
One issue with expressing cyclotides in different host plants is ensuring that the host plant has the necessary cellular machinery to process the expressed polypeptides to produce mature cyclic molecules. One approach is to identify host plant families that produce naturally occurring cyclotides, and to adapt the natural genes to facilitate the expression of foreign or engineered cyclotides (e.g., cyclotides from other plant families, or cyclotides engineered to contain one or more grafted peptide sequences). Until recently cyclotides had been found only in the Rubiaceae (coffee) and Violaceae (violet) plant families (Kaas, Q., Westermann, J. C. & Craik, D. J. (2010) Toxicon 55, 1491-509), apart from two atypical members in the Cucurbitaceae (cucurbit) family (Chiche, L., Heitz, A., Gelly, J. C., Gracy, J., Chau, P. T., Ha, P. T., Hernandez, J. F. & Le-Nguyen, D. (2004) Curr Protein Pept Sci 5, 341-349). Cyclotides from the Rubiaceae and Violaceae are biosynthesized via processing from dedicated precursor proteins encoded by multi-domain genes which contain one, two or three cyclotide domains (Dutton, J. L., Renda, R. F., Waine, C., Clark, R. J., Daly, N. L., Jennings, C. V., Anderson, M. A. & Craik, D. J. (2004) J. Biol. Chem. 279, 46858-46867).
There remains a need for expression systems configured to express foreign and modified cyclotides in additional plant families.